This invention relates to an improved nozzle apparatus for a flow cytometer system and methods for improving flow cytometry. Specifically, this invention relates to a novel design of a nozzle interior surface geometry that gently handles and orients a sample into a proper radial direction for analyzing and efficient sorting. The invention also focuses on systems for sorting delicate cells, especially living sperm cells.
Flow cytometers have been in clinical and research use for many years and their applications in animal industry such as animal breeding industry has been rapidly increasing. A commercially available flow cytometer typically utilizes a cylindrical fluid geometry in its nozzle. This type of flow cytometer system has a focusing flow path with symmetry of revolution, as described in some US Patents (U.S. Pat Nos. 5,602,039, 5,483,469, 4,660,971, 4,988,619 and 5,466,572). This type of design, according to the law of similarity, does not produce radially oriented samples. In clinical, animal breeding, and biological research fields, when cells such as sperm cells are sorted, they may be pre-stained with a dye that produces fluorescence when exposed to an excitation light source. As was explained in U.S. Pat. No. 5,135,759 to Lawrence Johnson, a flow cytometer that detects the emitted fluorescence perpendicular to the axis of flow can be used with high precision in the measurement and discrimination of the DNA content of the cells. However, as others have noted, even this precision in measuring the DNA content can only be achieved most efficiently when the cells of interest are spherical or cylindrical (Dean et al., 1978, Biophys. J. 23: 1-5). As for sperm cellsxe2x80x94which have flattened headsxe2x80x94the observed fluorescence intensity depends largely upon the proper orientation of the heads with respect to the detector. Sperm cells emit a stronger fluorescent signal from the edge than the flat surface Therefore, the intensity of the fluorescent signal is dependent on the orientation of the sperm head as it passes the detector. Because DNA content is determined by fluorescence and because fluorescent intensity is affected by orientation, DNA content determination can be compounded by lack of orientation in a nozzle. For this reason, without radial orientation, the resulting fluorescence intensity distribution obtained for normal, randomly oriented sperm heads reflects both DNA content and head orientation. Because the cells emit a brighter fluorescence signal from the head edge (Gledhill et al., 1976, J. Cell Physiol. 87: 367-376; Pinkel et al., 1982, Cytometry 7: 268-273) the accuracy of DNA content determination (which may differ by as little as 3.5%) is highly affected by the cells orientation. For this reason, the conventional flow cytometer has experienced limitations, especially when sorting flattened sperm cells or other non spherical or non-cylindrical cells and the like.
Additionally, certain cells can exhibit decreased functionality as a result of the sort process. This can be particularly true for cells such as mammalian sperm cells which are not only mechanically delicate, but also which can become functionally impaired (as perhaps seen through reduced fertility) or even mortally wounded as a result of some occurrence in the sort process. For flow cytometry efforts with delicate cells there have been significant limitations on abilities. This is most acute in the highly specialized field of sperm cell sorting not only because the cells themselves are unusually delicate, but also because there is a need for extremely high sorting rates for physiological and practical reasons. These two competing needs have proven to pose uniquely critical challenges in the unique field of sperm sorting for commercial breeding purposes. Thus, while these two aspectsxe2x80x94gentle handling and orientationxe2x80x94are perhaps independently applicable to a variety of instances, in many instances they can act synergistically. Both their independent characters and their synergistic interrelations are perhaps most acute in the commercial sperm sorting field. Interestingly, this synergy and potential interrelationship appears not to have been fully appreciated prior to the present invention.
Viewed in isolation, the aspect of proper orientation of a sample containing particles or cells can thus be seen to play an important role in the flow cytometer signal intensity and quality and in sorting efficiency. Efforts to hydrodynamically orient the sample have been made and the use of hydrodynamic orientation of the sample in flow through systems and flow cytometers have been explored in last few decades (Fulwyler, 1977, J. Histochem. Cytochem. 25: 781-783; Kachel et al., 1977, J. Histochem. Cytochem. 25: 774-780; Dean et al., supra). Hydrodynamic orientation of the sample within the flow cytometer can enhance precise measurement of relative DNA-stain content and can also provide a potentially useful measurement of morphological parameters such as cell thickness and degree of curvature of the flat face. For some applications, this orientation is straightforward. However, when delicate cells (such as sperm cells) or other particles are involved, however, a more gentle technique has been necessary. For example, a sample injection tube with a wedge shaped tip has even been used in some efforts to increase percentage of the oriented cells (Dean et al., 1978, Biophys. J. 23: 1-5; Fulwyler, 1977, J. Histochem. Cytochem. 25: 781-783; Johnson et al., 1986, Cytometry 7: 268-273; Pinkel et al., 1982, Cytometry 3: 1-9; Welch et al., 1994, Cytometry 17 (suppl. 7): 74). Because of the wedge shaped tip of the sample injection tube, the sample stream tended to be drawn into a thin ribbon by the sheath fluid as opposed to a cylindrical stream. Cells with flat heads such as mammalian sperm, often encountered the sheath fluid at a higher speed (100 mm/sec), and were then rotated so that their flat sides were in the plane of the ribbon. Unfortunately, the separation of the orientation event and the ultimate analysis event can cause less than optimal results. Therefore, this technique has not been practically shown to be as advantageous as desired.
In a different application, Kachel and his colleagues (Kachel et al., supra) demonstrated the law of similarity and discussed three types of flow paths that influenced the moving particles. They concluded that, to achieve uniform radial orientation with hydrodynamic forces for flat particles such as flattened red blood cells, the preferred flow path would be the one whereby unilateral constriction can be obtained. The most simple flow path that exhibits an increased unilateral constriction in use with a flow through system would be made of a tube with an ellipsoidal cross section, and would also end in an ellipsoidal outlet. In one arrangement, the long axis of this ellipsoidal outlet would be located at a right angle to the long axis in the cross section of the constricting elliptical tube. However, since the elliptical outlet does not produce the type of droplets desired for a high speed flow cytometer cell sorter, this arrangement was not intended to be used in, and has apparently not been applied to, a flow cytometer.
In a similar effort, Rens and his colleagues designed a nozzle tip that had an elliptical interior and an elliptical exit orifice (Rens et al., 1998, PCT Publication No. PCT/US98/15403; Rens et al., 1998, Cytometry 33: 476481; Rens et al., 1999, Mol. Reprod. Dev. 52: 50-56). This interior contained a first ellipsoidal zone and a second ellipsoidal zone that were separated by a transitional zone. All the zones each had a long axis and a short axis. The long axis of the second ellipsoidal zone was oriented 90xc2x0 to that of the first ellipsoidal zone. A cylindrical orifice, drilled through a jewel, was located at the end of the ellipsoidal exit orifice and served as the final exit. This device partially solved the problem of random orientation as existed in a conventional flow cytometer and could orient about 60% of the total flattened sperm cells from a boar each time through the flow cytometer. Nevertheless, when hydrodynamic forces in a flow path were taken into consideration, flat particles passing through the nozzle designed by Rens and his colleagues have received unnecessary stresses. For delicate cells, and especially for the perhaps more delicate sperm cells such as equine or bovine sperm cells, this approach simply did not appear to yield the desired efficiency either in orientation or in cell viability.
Thus, there existed a long felt but unsatisfied need for the invention while the needed implementing arts and elements had long been available. This need concerned the ability to gently handle and perhaps orient the particles or cells to be analyzed, the ability to properly analyze and sort efficiently, and the ability to minimize the potentially stressful situation that the flow cytometer caused for the particles or cells. Further, while problems existed in conventional flow cytometers, a full appreciation that a problem existed and what the problem was theretofore unseen by those skilled in the art. Substantial attempts by those skilled in the art to fill the need or to cope with the difficulties had been made but had not been fully successful most likely because of a failure to understand what exactly the problems were and perhaps how they interrelated. Some efforts made by those skilled in the art even matured into patents which seemed to have touched on the problems but, in fact, they tended in some regards to teach away from the technical direction in which the present inventors went.
It is therefore the object to present an improved nozzle interior surface geometry that produces the simplest flow path for applying necessary hydrodynamic forces to accelerate and perhaps orient a sample into a proper direction for analyzing and efficient sorting purposes. This improved nozzle interior surface geometry can comprise either or both of an appropriately configured accelerative force feature and/or an elliptical-like, single torsional interior surface element within a single torsional orientation nozzle that produces the special hydrodynamic forces, namely, single torsional orientation forces.
As the present invention now shows, the problems with undesirable cell stress could be viewed as at least in part due to either inappropriate handling forces, specifically: inappropriate accelerative forces, or the existence of a second torsional force created by the second ellipsoidal zone. As to the accelerative forces applied, devices often utilized abrupt transitions internal to the nozzle and so caused extreme acceleration over short distances. As to the orientation aspect, for example, approaches such as that of Ren (mentioned earlier) showed, that after cells had been oriented by a first torsional force created by a first ellipsoidal zone, an additionalxe2x80x94perhaps doublingxe2x80x94stress was applied. Specifically, the flat particles were already in an oriented position after they were oriented from a random position by the first ellipsoidal zone. They were ready to exit in oriented positions. At this time, however, the devices of Rens and others unnecessarily twisted these oriented flat particles a second time by the hydrodynamic forces created by a second ellipsoidal zone. As the present invention shows, these designs are not fully efficient in a high speed flow cytometer. When flat sperm cells with tails are oriented through this type of the nozzle, besides its inefficiency, the geometry in this type of the nozzle apparently impacts twice the torsional forces. This appears to unnecessarily and highly stress or damage the long tailed sperm cells before they exit the nozzle. In addition, in some designs where the orifice is made of a jewel that is separate from the main interior, a smooth laminar flow can also be affected to some degree. This could cause almost instantaneous acceleration and so could unnecessarily stress the cells and could affect the orientation of the already oriented sperm cells. Therefore, the approaches of Rens and other more recent efforts actually teach away from the more efficient, less accelerative and less torsional and smooth laminar interior surface of an embodiment of the present invention.
It is another object to design the simplest nozzle interior surface geometry that provides laminar flow surface and at the same time that reduces the distortion of the sample, especially of sperm cells.
Yet another object is to present a system which can more quickly and more accurately measure and sort the sample, especially delicate sperm cells in research and clinical use and in the animal insemination industry.
A further object to provide methods for improving orientation and sorting efficiency of the sample, especially the sperm cells in the flow cytometry for research and clinical use and animal insemination industry.
Naturally, further objects of the invention are disclosed throughout other areas of the specification and claims.